Project B - Inner Ear Hair Cells: Bundle Development and Regeneration Manfred Auer PROJECT SUMMARY/ABSTRACT The hair bundle is a precision organelle responsible for mechanoelectrical transduction in vertebrate animal hair cells in inner ear and lateral line sensory system. It consists of stereocilia that contain a crosslinked actin bundle, which is connected to the surrounding plasma membrane via unconventional myosins (and also radixin). Correlative quantitative proteomics and 3D tomographic studies of resin-embedded chick utricle tissue sections have revealed the macromolecular inventory and an only partially ordered 3D organization. This proposal focuses on vitrified whole-mount stereocilia samples using cryo-tomography, which ensures nearest- to-native sample preservation and eliminates the lengthy search for adequate section geometry e.g. for visualization of the adaptation machinery. We propose to complete our 3D analysis of the unstained actin core, localize and identify unconventional myosins through positive heavy metal staining cryo-tomography and template matching, as well as immunocytochemical labeling of permeabilized or demembranated vitrified whole-mount stereocilia. We propose to identify the different localized unconventional myosin using known differences in macromolecular dimensions, dimerization state, and preferred regional distribution. Last we will study hair bundles of regenerated hair cells in zebrafish lateral line neuromast, chick utricle sensory epithelia and blotted stereocilia and embryonic and induced pluripotent stem cell (ESC/iPSC)-derived hair cell-like cells. We will test the hypotheses that (1) the actin core is organized more like a gel rather than a paracrystal; (2) there is a defined ultrastructural correlate of the predicted MYO1C-based adaptation machinery; (3) the different unconventional myosins residing in the hair bundle occupy different territories in stereocilia using whole-mount immuno-affinity labeling and heavy metal staining or prior to vitrification and cryo-EM imaging; (4) the hair bundle and stereocilia organization of regenerated hair cells ressembles those of untreated chick utricle and zebrafish lateral line hair bundles/stereocilia. We believe that our ultrastructural and affinity-labeling approaches will help settle a number of questions in the hair cell field, e.g. regarding actin organization and turnover, as well as unconventional myosin localization and function. Also, it will provide an ultrastructural framework in which to interpret other types of data with respect to hair cell regeneration. Last but not least our proposal provides a strong technical platform for future ultrastructural hair cell studies and beyond for the field of structural cell biology.